It is known that a large variety of naturally occurring, biologically active polypeptides bind heparin. Such heparin-binding polypeptides include cytokines, such as platelet factor 4 and IL-8 (Barber et al., (1972) Biochim. Biophys. Acta, 286:312-329; Handin et al., (1976) J. Biol. Chem., 251:4273-422; Loscalzo et al., (1985) Arch. Biochem. Biophys. 240:446-455; Zucker et al., (1989) Proc. Natl. Acad. Sci. USA, 86:7571-7574; Talpas et al., (1991) Biochim. Biophys. Acta, 1078:208-218; Webb et al., (1993) Proc. Natl. Acad. Sci. USA, 90:7158-7162) heparin-binding growth factors (Burgess and Maciag, (1989) Annu. Rev. Biochem., 58:576-606; Klagsbrun, (1989) Prog. Growth Factor Res., 1:207-235), such as epidermal growth factor (EGF); platelet-derived growth factor (PDGF); basic fibroblast growth factor (bFGF); acidic fibroblast growth factor (aFGF); vascular endothelial growth factor (VEGF); and hepatocyte growth factor (HGF) (Liu et al., (1992) Gastrointest. Liver Physiol. 26:G642-G649); and selectins, such as L-selectin, E-selectin and P-selectin (Norgard-Sumnicht et al., (1993) Science, 261:480-483). See also, Munoz and Linhardt., (2004) Arterioscler Thromb Vasc Biol., 24:1549-1557.
International Publication No. WO 95/07097 describes formulations of heparin binding proteins including heparin binding growth factors such as VEGF, with purified native heparin or other polyanionic compounds for therapeutic use. Heparin derived oligosaccharides and various other polyanionic compounds have been shown to stabilize the active conformation for heparin binding growth factors (Barzu et al., (1989) J. Cell. Physiol. 140:538-548; Dabora et al., (1991) J. Biol. Chem. 266:23627-23640) and heparin affinity chromatography has been employed in various purification schemes (see generally, International Publication No. WO 96/02562).
Many of the heparin binding proteins of mammalian origin have been produced by recombinant technology and are clinically relevant (Munoz and Linhardt, (2004) Arterioscler Thromb Vasc Biol., 24:1549-1557; Favard et al. (1996) Diabetes and Metabolism 22(4):268-73; Matsuda et al., (1995) J. Biochem. 118(3):643-9; Roberts et al., (1995) Brain Research 699(1):51-61). For example, VEGF is a potent mitogen for vascular endothelial cells. It is also known as vascular permeability factor (VPF). See, Dvorak et al., (1995) Am. J. Pathol. 146:1029-39. VEGF play important roles in both vasculogenesis, the development of the embryonic vasculature, and angiogenesis, the process of forming new blood vessels from pre-existing ones. See, e.g., Ferrara, (2004) Endocrine Reviews 25(4):581-611; Risau et al., (1988) Dev. Biol., 125:441-450; Zachary, (1998) Intl. J. Biochem Cell Bio 30:1169-1174; Neufeld et al., (1999) FASEB J. 13:9-22; Ferrara (1999) J. Mol. Med. 77:527-543; and, Ferrara and Davis-Smyth, (1997) Endocri. Rev. 18:4-25. Clinical applications for VEGF include those where the growth of new capillary beds is indicated as, for example, in promoting wound healing (see, for example, International Publication No. WO 91/02058; and, Ser. No. 11/455,017, entitled “Wound Healing” filed on Jun. 16, 2006), in promoting tissue growth and repair, e.g., liver (see, e.g., WO2003/0103581), bone (see, e.g., WO2003/094617), etc. See also, Ferrara, (2004) Endocrine Reviews 25(4):581-611.
Typically, therapeutically relevant recombinant proteins are produced in a variety of host organisms. Most proteins can be expressed in their native form in eukaryotic hosts such as CHO cells. Animal cell culture generally requires prolonged growing times to achieve maximum cell density and ultimately achieves lower cell density than prokaryotic cell cultures (Cleland, J. (1993) ACS Symposium Series 526, Protein Folding: In Vivo and In Vitro, American Chemical Society). Additionally, animal cell cultures often require expensive media containing growth components that may interfere with the recovery of the desired protein. Bacterial host expression systems provide a cost-effective alternative to the manufacturing scale production of recombinant proteins. Numerous U.S. patents on general bacterial expression of recombinant proteins exist, including U.S. Pat. Nos. 4,565,785; 4,673,641; 4,795,706; and 4,710,473. A major advantage of the production method is the ability to easily isolate the product from the cellular components by centrifugation or microfiltration. See, e.g., Kipriyanov and Little, (1999) Molecular Biotechnology, 12: 173-201; and, Skerra and Pluckthun, (1988) Science, 240: 1038-1040.
Recombinant heparin binding growth factors such as acidic fibroblast growth factor, basic fibroblast growth factor and vascular endothelial growth factor have been recovered and purified from a number of sources including bacteria (Salter D. H. et al., (1996) Labor. Invest. 74(2):546-556 (VEGF); Siemeister et al., (1996) Biochem. Biophys. Res. Commun. 222(2):249-55 (VEGF); Cao et al., (1996) J. Biol. Chem. 261(6):3154-62 (VEGF); Yang et al., (1994) Gaojishu Tongxun, 4:28-31 (VEGF); Anspach et al., (1995) J. Chromatogr. A 711(1):129-139 (aFGF and bFGF); Gaulandris (1994) J. Cell. Physiol. 161(1):149-59 (bFGF); Estape and Rinas (1996) Biotech. Tech. 10(7):481-484 (bFGF); McDonald et al., (1995) FASEB J. 9(3):A410 (bFGF)). However, bacterial expression systems such as E. coli lack the cellular machinery to facilitate proper refolding of the proteins and generally do not result in the secretion of large proteins into the culture media. Recombinant proteins expressed in bacterial host cells are often found as inclusion bodies consisting of dense masses of partially folded and misfolded reduced protein. In this form, the recombinant protein is generally inactive. For example, the predominant active form of VEGF is a homodimer of two 165-amino acid polypeptides (VEGF-165). In this structure, each subunit contains 7 pairs of intrachain disulfide bonds and two additional pairs which effect the covalent linkage of the two subunits (Ferrara et al., (1991) J. Cell. Biochem. 47:211-218). The native conformation includes a strongly basic domain which has been shown to readily bind heparin (Ferrara et al (1991) supra). Covalent dimerization of VEGF is needed for effective receptor binding and biological activity (Pötgens et al., (1994) J. Biol. Chem. 269:32879-32885; Claffey et al., (1995) Biochim. et Biophys. Acta 1246:1-9). The bacterial product potentially contains several misfolded and disulfide scrambled intermediates.
Additionally, refolding often produces misfolded and disulfide-linked dimers, trimers, and multimers. (Morris et al., (1990) Biochem. J., 268:803-806; Toren et al., (1988) Anal. Biochem., 169:287-299). This association phenomenon is very common during protein refolding, particularly at higher protein concentrations, and appears often to involve association through hydrophobic interaction of partially folded intermediates (Cleland and Wang, (1990) Biochemistry, 29:11072-11078).
Misfolding occurs either in the cell during fermentation or during the isolation procedure. Proteins recovered from periplasmic or intracellular space must be solubilized and the soluble protein refolded into the native state. In vitro methods for refolding the proteins into the correct, biologically active conformation are essential for obtaining functional proteins. Typical downstream processing of proteins recovered from inclusion bodies includes the dissolution of the inclusion body at high concentration of a denaturant such as urea followed by dilution of the denaturant to permit refolding to occur (see, U.S. Pat. Nos. 4,512,922; 4,511,502; and 4,511,503). See also, e.g., Rudolph and Lilie, (1996) FASEB J. 10:49-56; and, Fischer et al., (1993), Biotechnology and Bioengineering, 41:3-13. Such recovery methods are regarded as being universally applicable, with minor modifications, to the recovery of biologically active recombinant proteins from inclusion bodies. These methods have been applied to heparin binding protein such as VEGF (Siemeister et al. (1996) supra). These methods seek to eliminate random disulfide bonding prior to coaxing the recombinant protein into its biologically active conformation through its other stabilizing forces and may not eliminate improperly folded intermediates or provide homogenous populations of properly folded product.
Reversed micelles or ion exchange chromatography have been used to assist refolding of denatured proteins by enclosing a single protein within micelles or isolating them on a resin and then removing the denaturant (Hagen et al., (1990) Biotechnol. Bioeng. 35:966-975; Creighton (1985) in Protein Structure Folding and Design (Oxender, D. L. Ed.) pp. 249-251, New York: Alan R. Liss, Inc.). These methods have been useful in preventing protein aggregation and facilitating proper refolding. To alter the rate or extent of refolding, conformation-specific refolding has been performed with ligands and antibodies to the native structure of the protein (Cleland and Wang, (1993), in Biotechnology, (Rehm H.-J., and Reed G. Eds.) pp 528-555, New York, VCH). For example, creatine kinase was refolded in the presence of antibodies to the native structure (Morris et al., (1987) Biochem. J. 248:53-57). In addition to antibodies, ligands and cofactors have been used to enhance refolding. These molecules would be more likely to interact with the folding protein after formation of the native protein. Therefore, the folding equilibrium could be “driven” to the native state. For example, the rate of refolding of ferricytochrome c was enhanced by the extrinsic ligand for the axial position of the heme iron (Brems and Stellwagon, (1983) J. Biol. Chem. 258:3655-3661). Chaperone proteins have also been used to assist with protein folding. See, e.g., Baneyx, (1999) Current Opinion in Biotechnology, 10:411-421.
There is a need for new and more effective methods of folding and/or recovering heparin binding proteins from a host cell culture, e.g., for the efficient and economical production of heparin binding proteins in bacterial cell culture that provides for elimination or reduction of biologically inactive intermediates and improved recovery of a highly purified biologically active properly refolded protein and that is generally applicable to manufacturing scale production of the proteins. The invention addresses these and other needs, as will be apparent upon review of the following disclosure.